The major objective of this project is to describe the cellular mechanisms by which hormones, neurotransmitters and paracrines interact to control hydrogen ion secretion by parietal cells in the gastric mucosa. The experimental models that will be utilized include acutely isolated gastric glands and highly enriched (95% pure) parietal cell preparations. Studies designed to develop a technique for culturing homogeneous, hormonally responsive parietal cells will be continued. The various cellular preparations will be used to study receptor mechanisms, the roles of calcium and cyclic nucleotides in stimulus-secretion coupling, cAMP-dependent and independent protein kinase activities as well as to identify and characterize phosphorylated intermediates involved in the regulation of the acid secretroy process. Enzymes and inositol phosphates will be isolated using HPLC and/or FPLC (fast protein liquid chromatography). Phosphorylated proteins will be identified in parietal cell extracts using two dimensional gel electrophoresis (IEF. SDS-PAGE), antibody precipitation, autoradiography, protein blotting, and FPLC. Changes in free intracellular calcium will be assessed with recently synthesized fluorescent calcium probes, such as fura 2, using a spectrofluorimeter interfaced with a lab computer and digitized video image analysis. Redox changes in pyridine nucleotides will also be measured by these techniques. Biochemical responses will be measured in conjunction with previously established indices of acid secretory responsiveness including cellular respiration, aminopyrine uptake and morphological transformations. The characterization and correlation of both physiological and biochemical parameters will provide a basis for determining the sequence of events that occur following secretagogue-receptor interaction that ultimately lead to activation of the parietal cell hydrogen ion "pump". Through comparisons of activities in enriched parietal cells, cultured parietal cells and gastric glands, which are composed of several cell types including endocrine-like cells, significant basic information will be obtained that may ultimately provide for more enlightened clinical evaluation and treatment of peptic ulcer disease.